Changes in cytokine expression in peripheral leukocytes, detected by microarray analysis, in the secretory vs proliferative phase of the menstrual cycle

Abstract

Objective: Many autoimmune disorders have recognized clinical changes in pregnancy and the menstrual cycle. However the mechanisms by which hormones, menstrual cycle changes and pregnancy affect the immune system in unknown. The complexity of the immune system and abundance of immune markers necessitate global analysis. Herein, we describe high density oligonucleotide microarray profiling of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Design: Microarray analysis of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Materials/Methods: Peripheral blood was obtained from healthy female volunteers (n=5) after informed consent. Subjects were 25–34 yo with menstrual cycles of 28–30 days. Paired samples were collected from subjects in nonconception cycles in the late proliferative (cycle day 10–12) and midsecretory phase (day 20–24). Total RNA was extracted using the Paxgene Blood RNA Validation Kit. cDNA was synthesized from total RNA then biotinylated cRNAs were generated by in vitro transcription. Fragmented cRNAs were hybridized on Affymetrix HU95A microarrays, containing 12,686 human genes and ESTs. The data were analyzed with GeneChip Analysis Suite v5.0, GeneSpring v4.2& MS Excel. Results from each phase were normalized and gene expression was compared. Non-parametric testing was applied to genes showing 1.5 fold changes between the proliferative and secretory phases with a p <0.05 for statistical significance. Results: 10 cc of blood yielded 10–15 g of leukocyte RNA. 17 g of high purity, labeled cRNA was used for hybridization. 314 genes were found to be significantly up-regulated in the secretory phase compared to proliferative, including NKG2F, complement receptor C1r, fibroblast growth factor, monocyte chemotactic protein-2, C-C chemokine receptor-4, platelet derived growth factor receptor and natural killer cell stimulatory factor (IL 12). 362 genes were significantly down-regulated in the secretory phase, such as allograft inflammatory factor-1(AIF-1), pre-B cell enhancing factor (PBEF), GM-CSF receptor beta chain, leukocyte immunoglobulin-like receptor-3(LIR-3), interferon regulatory factor 1, lymphocyte activation antigen CD30, and pT49. Conclusions: We have shown significant changes in the expression of cytokines and their receptors in the menstrual cycle. While validation of these findings and further study are needed to determine their clinical significance, immune cells and their effector cytokines are actively involved in the process of implantation and maternal tolerance to a developing fetus. Peripheral changes in immune cells have been previously described in patients with recurrent spontaneous abortion and unexplained infertility. This study lays foundation for further deliniation of steroid hormone control of immune function and dysfunction in women. Supported by: NIH Training grant HD07493 and Women’s Health at Stanford.